KR20050028018A - Novel chemical substance having activity for morphogenesis and growth promotion - Google Patents

Abstract

A novel chemical substance capable of inducing the morphogenesis and growth promotion of marine large green algae; a process for producing the novel chemical substance with the use of a microorganism; and an algae culture medium comprising the novel chemical substance.

Description

Novel Chemical Substance Having Activity for Morphogenesis and Growth Promotion

The present invention relates to a novel vitamin-like active substance produced by microorganisms and a method and use of the same.

Until now, indoor cultures using existing synthetic mediums of large green algae such as brown and unripe algae have become callus phases depending on the differentiation induction after dedifferentiation, and the body of the algae collapses in a single cell state. (L. Provasoli; Ulva. Biol. Bull., 1958, 114, 375: M. Tatewaki, L. Provasoli and IJ Pintner; J. Phycol., 1983, 19, 409). It is also known that addition of fresh seawater, soil extracts, flushing extracts, brown algae extracts, or marine species-derived microbial cultures to these lost algae results in the recovery of thallus form or increased growth rates (see : M. Tatewaki, L. Provasoli and IJ Pintner; J. Phycol., 1983, 19, 409), no active ingredients or vitamin-like active substances of these extracts have been identified. As a result, long-term physiological, ecological studies or culture preservation using sterile cultured cells of large green algae, such as brown or single green, is difficult.

1 shows a mass spectrum of novel compound 1.

2 shows the ¹ H-NMR spectrum of novel compound 1. FIG.

3 shows the ¹³ C-NMR spectrum of New Compound 1.

4 shows the ¹ H-NMR spectrum of novel compound 2.

5 is a photograph of Monostroma oxyspermum culture cells after 5 days of culture when no sample was added in the Monostroma oxyspermum culture experiment of Example 1. FIG.

FIG. 6 is a photograph of monostroma oxyspermum cultured cells 5 days after the culture when the sample is added in the monostroma oxyspermum culture experiment of Example 1. FIG.

7 shows the ¹ H-NMR spectrum of Me1.

8 shows the ¹³ C-NMR spectrum of Me1.

9 shows the ¹ H-NMR spectrum of Me1B.

10 shows the ¹ H-NMR spectrum of Me1H3.

11 shows the ¹ H-NMR spectrum of Me1H1.

12 shows the ¹ H-NMR spectrum of Me ¹ H ¹ Me.

Figure 13 is a photograph showing the results of the seven-step dilution 10 days after the culture of Example 5.

Figure 14 is a photograph showing the results of the six-step dilution 10 days after the culture of Example 5.

15 is a photograph showing the experimental results of Ulva pertusa of Example 7.

16 is a photograph showing the experimental results of Enteromorpha interstinalis of Example 7.

17 shows the gyrB DNA lineage of the YM-1-69 strain, YM-2-23 strain, and similar strains thereof.

FIG. 18 shows the 16S rDNA lineage of YM-1-69 strain, YM-2-23 strain and similar strains, and the specific activity of morphogenesis induction for large green algae of these strains.

19 shows a partial structure of novel compound 1.

20 shows the results of analyzing the HBMC spectrum of novel compound 1. FIG.

21 shows the substructure and relative stereoconfiguration of Me1.

22 shows the result of analyzing the HBMC spectrum of Me1.

Fig. 23 shows the results of analyzing the spectrum of Me1H3.

24 shows the result of analyzing the HMBC spectrum of Me1H3.

25 shows the result of analyzing the spectrum of Me1H1.

Fig. 26 shows the results of analyzing the HBMC spectrum of Me1H1.

Fig. 27 shows the results of analyzing the spectrum of Me1H1Me.

Fig. 28 shows the results of analyzing the HBMC spectrum of Me1H1Me.

This specification includes content described in the specification and / or drawings of 2002-203608, which is a priority document of the present application.

Disclosure of the Invention

If it is possible to cultivate marine large green algae aseptically for a long period of time using only a medium already known in its entirety, in addition to the physiological and ecological studies of algae to be cultured, it may be possible to produce or maintain seedlings of cultivated edible seaweeds. It can be established by practical culture methods such as management and preservation of green algae species. From this point of view, the present inventors have already identified microorganisms that induce morphogenesis and growth promotion of marine large green algae from this point of view (Special Application 2001-396342), but the vitamin-like active substance that is an active ingredient produced by the microorganism is unknown. to be.

The present invention aims to provide such novel vitamin-like active substances.

Accordingly, the present inventors have searched for a novel active ingredient, YM-2-23 (accession number FERM BP-8417, August 20, 2001 (original deposit) to the Institute of Industrial Technology Research Institute of Korea Institute of Industrial Technology Patent Biology Deposit) Deposited in the Center (1-1, 1-1 Higashi 1-chome, Tsukubashi, Ibaragiken, Japan) (Received the request for transfer by deposit in accordance with the Budapest Treaty from the original deposit on 25 June 2003), Tenashi Baculum species ( Tenacibaculum sp.) YM-1-69 (Accession No. FERM BP-8418, dated August 20, 2001) (Original Deposit) Patent Biodeposit Center, Industrial Technology Research Institute, Independent Administrative Institution (Higashi Ibarakiken, Tsukuba City, Japan) Deposited on Chome 1-1, Chuodai 6) (receipt of the request for transfer from the deposit in accordance with the Budapest Treaty from the original deposit on 25 June 2003) and the active substance having very strong activity from the culture of the similar strains As a result of isolation, it was confirmed that the electric compound was a new substance, You have completed the command. To date, lycopene is a substance produced by Flavobacterium , Zobellia or Tenacibaculum which is similar to YM-2-23 and / or YM-1-69. Although carotenoid derivatives, such as zeaxanthin, have been reported, there are no examples of isolating compounds that promote growth or modulate morphology of large algae, and are the first to be reported in the world.

That is, this invention includes the following inventions.

(1) Compound 1 having the following physicochemical properties:

1. Color of material: colorless

2. Molecular Weight: 457

3. Molecular Formula: C ₂₄ H ₃₁ N ₃ O ₄ S

Mass spectrometry: FABMS: m / z 456 [M ⁻ H] ⁻ (FIG. 1)

High resolution mass spectrometry: found 456.1960 [MH] ^-

Calculated 456.1930 (C ₂₄ H ₃₁ N ₃ O ₄ S)

4. Nuclear magnetic resonance signal:

1) ¹ H-NMR (D ₂ O-20 mM Na ₂ HPO ₄ (pH 9), 750 MHz): (FIG. 2)

δppm 0.818 (3H, s), 0.837 (3H, s), 0.882 (3H, s), 0.960 (1H, m), 1.058 (1H, m), 1.167 (1H, m), 1.326 (3H, s), 1.37 (1H, m), 1.38 (1H, m), 1.40 (1H, m), 1.58 (1H, m), 1.61 (2H, m), 1.52 (1H, br d, J = 13 Hz), 1.76 (1H , br d, J = 14 Hz), 2.024 (1H, m), 2.181 (1H, dd, J = 4, 14 Hz), 2.291 (1H, dd, J = 14, 16.5 Hz), 7.698 (1H, d, J = 7.5 Hz), 7.845 (1H, d, J = 7.5 Hz)

2) ¹³ C-NMR (D ₂ O-20 mM Na ₂ HPO ₄ (pH 9), 125 MHz): (FIG. 3)

δ ppm 15.236 (q), 19.037 (t), 20.287 (t), 20.955 (q), 21.835 (q), 25.987 (t), 33.381 (s), 33.636 (q), 37.308 (s), 39.590 (t) , 41.199 (t), 42.346 (t), 52.769 (d), 56.381 (d), 79.096 (s), 114.965 (s), 124.399 (d), 139.004 (s), 141.232 (d), 150.282 (s) , 152.656 (s), 172.081 (s), 173.538 (s), 174.661 (s).

 (2) Compound 2 having the following physicochemical properties:

1. Color of material: colorless

2. Nuclear magnetic resonance signal:

¹ H-NMR (D ₂ O-20 mM Na ₂ HPO ₄ (pH 9), 500 MHz): (Figure 4)

δ ppm 0.815 (3H, s), 0.834 (3H, s), 0.877 (3H, s), 0.949 (1H, m), 1.048 (1H, m), 1.163 (1H, m), 1.297 (3H, s), 1.35-1.40 (3H, m), 1.52-1.63 (4H, m), 1.753 (1H, br d, J = 14 Hz), 2.012 (1H, m), 2.158 (1H, m), 2.299 (1H, m) , 7.646 (1H, d, J = 8.0 Hz), 7.769 (1H, d, J = 8.0 Hz).

(3) Microorganisms having the ability to produce Compound 1 or Compound 2 in electricity (1) above are cultured in a medium, and Compound 1 or 2 is produced and accumulated in the culture to generate electricity and accumulate. Process for the preparation of compound 1 or 2, characterized in that compound 1 or 2 is obtained.

(4) The method of producing the former (3), wherein the microorganism is YM-2-23 strain (FERM BP-8417), Tenacibaculum sp., YM-1-69 (FERM BP-8418) or similar strains thereof. .

(5) A medium for algae culture comprising compound 1 of item (1) or compound 2 of item (2) as an active ingredient.

(6) The monomethylated, dimethylated or trimethylated compound of Compound 1 obtained by treating Compound 1 in (1) with trimethylsilyldiazomethane.

(7) A compound obtained by treating the trimethylated substance of (6) with sodium borohydride or a derivative thereof.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Compounds 1 and 2 of the present invention can be produced using microorganisms. The microorganism used in the preparation of the compound of the present invention is not particularly limited as long as it is a microorganism having a production capacity of the electric compound, for example, to Cytophaga-Flavobacterium-Bacteriodes complex, such as Flavobacterium , Gibelia, Tenassivaculum and the like. And strains derived from these strains or these strains. Specifically, mutant strains derived from YM-2-23 strain (FERM BP-8417), Tenacibaculum sp., YM-1-69, or strains thereof are exemplified. Instead of YM-1-69 or YM-2-23, similar strains of these strains may be used. Examples of "similar strains of YM-1-69 strain" include, for example, a strain showing a frond forming activity or growth promoting activity against marine large green alga, and at least 85%, preferably at least 95% homologous to the nucleotide sequence of SEQ ID NO: 1. Strains having a 16S rRNA V3 region gene having one nucleotide sequence or a gyrB gene having a nucleotide sequence of at least 72%, preferably at least 95%, are homologous to the nucleotide sequence of SEQ ID NO: 3. "YM-2-23 strain-like strain" is, for example, a strain showing a frond forming activity or growth promoting activity against marine large green algae, at least 85%, preferably at least 95% homologous to the nucleotide sequence of SEQ ID NO: 2 A strain having a 16S rRNA V3 region gene having one nucleotide sequence or a gyrB gene having a nucleotide sequence which is at least 72%, preferably at least 80%, more preferably at least 95% homologous to a nucleotide sequence of SEQ ID NO: 4 Strains are included.

Examples of "similar strains of YM-1-69 strain" and "similar strains of strain YM-2-23" include, for example, YM2-10 (MBIC 04671), YM2-11 (MBIC 04672), YM2-12 (MBIC 04673), YM2-13 (MBIC 04674), YM1-66 (MBIC 04663), YM2-24 (MBIC 04684), YM1-51 (MBIC 04662), Zobellia uliginosa (ATCC 14397), YM1-11 (MBIC 04693), T-588 (MBIC 05930), YM2-22 (MBIC 04682), YM2-27 (MBIC 04687), YM2-6 (MBIC 04669), YM1-68 (MBIC 04664), YM1-38 (MBIC 04661), YM2-4 (MBIC 04667), YM2-5 (MBIC 04668), YM2-7 (MBIC 04670), YM2-21 (MBIC 04681), YM2-1 (MBIC 04666), T-565 (MBIC 05877), T-424 (MBIC 05876) , Cytophaga sp. UP7 (MBIC 01484), T-551 (MBIC 05929), Pedobacter heparinus (IFO 12017), T-561 (MBIC 05879), Cyclobacterium marinum (LMG 13164), Cytophaga sp. (MBIC 01539), Cytophaga sp. (MBIC 01599), Chitinophaga pinensis (DSM 2588).

Among the above strains, strains described with "MBIC" include Marine Biotechnology Institute Culture Collection (MBIC, 3-75-1 Heita, Kamaishi City, Iwateken, Japan) (http: //seasquirt.mbio In .co.jp / mbic / index.php? page = top, the strains listed with "IFO" are located at the Institute for Fermentation (OFO, IFO, 17 Hocho-machi 2-chome, Yodogawa-ku, Osaka-fu, Japan). -85), the strains described "ATCC" is from the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852, USA), the strains described "DSM" is Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH : In DSMZ, Mascheroder Weg 1b, 38124 Braunschweig, Germany), strains in which "LMG" is described are BCCM ^TM / LMG Bacteria Collection (Belgian Co-ordinated Collections of Micro-organisms, Laboratorium voor Microbiologie, Universiteit Gent (RUG), KL Ledegancksfaat) 35, B-9000 G ent, Brussels, Belgium).

Strains having the same nucleotide sequence of the 16S rRNA V3 region gene and / or the gyrB gene as the nucleotide sequences of the YM-1-69 strain or the YM-2-23 strain are particularly preferred strains among these similar strains.

When culturing the electric microorganisms, a conventional marine bacterial culture method is generally used. As the medium, any medium can be used, as long as it is a medium containing a magnetizable carbon source, a nitrogen source, an inorganic substance, and the like.

As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol and molasses can be used alone or in combination.

Nitrogen sources include ammonium chloride, ammonium nitrate, ammonium sulfate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soy flour, casamino acid, etc. It can be used in combination.

In addition, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, magnesium phosphate octahydrate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, and copper sulfate are added as necessary.

In addition, a trace component (for example, sugars, amino acids, inorganic salts, etc.) that promotes growth of used bacteria or production of the compound of the present invention can be appropriately added.

As the culture method, the liquid culture is most efficient, the culture temperature is about 30 ℃ suitable, the pH of the medium is usually 7 to 9, preferably 7.5 to 8. Sodium hydroxide aqueous solution, hydrochloric acid, etc. can be used for pH adjustment of a medium.

When the culture is performed for 1 to 4 days in liquid culture, compound 1 or 2 is produced and accumulated in the culture medium and the cells. The culture is preferably stopped at the time when the amount of production in the culture reaches the maximum, and most preferably, three days after the culture.

When isolating compound 1 or 2 in a culture, conventional methods are used to isolate and purify microbial metabolites from the culture. For example, the culture is divided into culture filtrate and cells by filtration or centrifugation, and the cells are extracted with hydrous methanol, hydrous acetonitrile and the like. Subsequently, the organic solvent is removed from the extract by a rotary evaporator under reduced pressure, and the extract and the culture filtrate are combined and adsorbed with a polystyrene-based adsorbent (for example, styrenedivinylbenzene polymer). The adsorbed active substance is washed with water and desalted to elute the active substance with hydrous methanol, hydrous acetonitrile and the like. The eluate is concentrated under reduced pressure by lyophilization and the like to obtain Compound 1 or 2 by styrene divinylbenzene-based polymer, ion exchange chromatography with anion exchange resin, gel filtration chromatography, high performance liquid chromatography, and the like. The novel compound 2 is a very unstable compound, but when the new compound 2 is placed in a strong alkali aqueous solution such as aqueous sodium hydroxide solution or ammonia water, it gradually changes to new compound 1. New Compound 1 is a very stable compound.

Treatment of novel compound 1 with a suitable methylating agent (eg trimethylsilyldiazomethane) can yield the monomethylated, dimethylated or trimethylated compounds of novel compound 1. Monomethylation by adjusting the molar ratio of trimethylsilyldiazomethane to novel compound 1 or by selecting reaction conditions (e.g., setting the reaction temperature or pH, reaction in the presence of dimethylaminosulfurtrifluoride (DAST)) Sieve, dimethylated form or trimethylated form can optionally be obtained.

Further, when the trimethylated body is treated with sodium borohydride, Me1H3 having the following physicochemical properties can be obtained. In addition, by changing the polarity of the reaction solvent and the reaction temperature, it is possible to obtain a compound Me1H1 which is further reduced than Me1H3. In addition, Me1H1 is reacted with alkyl iodide (e.g., methyl iodide) represented by the formula RI (here, R represents an alkyl group having 1 to 6 carbon atoms) under strong alkali conditions to form an alkylated compound such as methylated Me1H1Me. Can be obtained. The information of these reactions and the obtained products is useful for specifying the novel compounds 1 and 2 and the like.

The novel compounds 1 and 2 of the present invention are useful as an active ingredient in algal culture medium. The novel compounds 1 and 2 may be used independently and may use both together.

As algae to be applied to the medium for electric algae culture, marine large green algae are preferable. Large marine algae are marine algae of the ULVALES family, and include, for example, algae such as Monostromataceae and Ulvaaceae. Specifically, examples of the seaweeds belonging to a single family include Monostroma nitidum , Monostroma oxyspermum , Monostroma angicava , Seaweeds belonging to a green sea family Enteromorpha compressa , Enteromorpha intestinalis , Enteromorpha linza , Ulva conglobata , Ulva pertusa and the like.

The medium used for cultivation of marine large green algae includes a medium similar to that used in conventional culture methods (culture method in which marine large green algae are single-celled), except that it contains novel compounds 1 and / or 2 as active ingredients. For example, ASP7 medium, PES medium, PESI medium, or the like, or simply sterile seawater can be used. The effective concentration of novel compounds 1 and / or 2 in the medium is not particularly limited as long as it is within a range capable of inducing frond formation, but is preferably set to 10 ⁻¹² to 10 ⁻³ μg / ml.

The temperature at the time of cultivation is not particularly limited as long as it is within the range in which marine large green algae can survive, but is preferably about 15 to 25 ° C.

Best Mode for Carrying Out the Invention

The present invention will be described in detail by way of examples. However, the technical scope of the present invention is not limited by these examples.

Reference Example 1 : Isolation of Microorganisms

The strain used in the present invention was isolated as follows. To about 1 g of collected fresh algae, 10 ml of sterilized seawater was added and stirred vigorously for about 1 minute. The supernatant was diluted 10-fold and 100-fold again with sterile seawater, 100 μl of which was dispensed on a 1/10 marine agar plate, and inoculated throughout the plate with a sterile Conradi stick. Sulfur to red colonies grown after 2-3 days at room temperature were inoculated into different marine agar plates and incubated until single colonies were isolated. 2 to 1 ml of ASP7 medium was dispensed into 24-48 well microplates, and about 20 cells of monostroma oxyspermum , which were monocellularized , were added to each well. Colonies of the isolates, which were homogenized, were directly inoculated in two wells using a sterile platinum loop or the like. The plate was incubated for 5 days at 19-22 ° C., light period 14 hours / dark period 10 hours, and frond formation of Monostroma oxyspermum was confirmed by an inverted microscope. Strains showing the formation of fronds were confirmed by further experiments in the same manner as before. As a result of the above screening, YM-1-69 strain and YM-2-23 strain were isolated as strains showing frond forming activity. The YM-1-69 strain is a strain isolated from Halimeda opuntia (Codiales, Chlorophyta) and the YM-2-23 strain is a strain isolated from Monostroma nitidum (Ulvales, Chlorophyta).

Reference Example 2 Identification of Microorganisms

The DNA sequences of the 16S rRNA V3 region and gyrB gene of the microorganism obtained in Reference Example 1 were determined. The DNA sequencing of the 16S rRNA V3 region of the YM-1-69 strain and the gyrB gene was shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. In addition, the DNA sequencing of the 16S rRNA V3 region of the YM-2-23 strain and the gyrB gene was shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. As a result of performing a database search (DDBJ-fasta) on the obtained sequence, each sequence showed high homology with the sequence of the microorganism shown in Table 1.

Table 1

order Homology (%) Creatures with related sequences Accession Number One 95.26 Tenacibaculum amylolyticum AB032505 2 94.18 Zobellia uliginosa M62799 3 84.91 Tenacibaculum amylolyticum AB032586 4 78.18 Zobellia uliginosa AB034224

In addition, the results of the investigation of the physiological and biochemical properties of YM-1-69 strain and YM-2-23 strain are shown in Table 2.

TABLE 2

Physiological and Biochemical Properties YM-1-69 YM-2-23 Gram staining - - Catalase activity + + Oxidase activity + + OF Test O F Request of magnesium or calcium + + Reduction of nitrate + + Indole production - - Gelatin hydrolysis - - Starch hydrolysis + + DNA hydrolysis + + Tween 80 Hydrolysis + + Esculin hydrolysis + + Arginine Dehydrolase Activity - - Urease activity - - β-galactosidase activity - + Use Simmons badge of citric acid - - Christensen badge - -

+: Positive,-: negative

From the results of Table 1 and Table 2, YM-1-69 strain was identified as Tenacibaculum sp., And YM-2-23 strain was identified as strain belonging to the Cytophaga-Flavobacterium-Bacteriodes complex.

Example 1 Monostroma oxyspermum culture experiment

Monostroma oxyspermum, which had been sterilized with antibiotics, did not show the lobular shape seen in natural state in the synthetic medium such as ASP7 medium and became almost unicellular.

1 ml of modified ASP7 medium is added to the first row of 48-well microplates (IWAKI Co., Ltd. abbreviated to 48-MTP), the second column and 0.9 ml are added thereto, and the first column is 11 μl of the active substance fraction to be tested. 100 μl was added to the next column with gentle addition and stirring well by pipetting. This operation could be repeated 8 to 18 times for each fraction to make 10-fold dilutions in 8 to 18 steps. 100 μl remaining in the last row was discarded. Here, 100 µl of Monostroma oxyspermum culture solution diluted to a final cell concentration of 10 to 20 cells per ml was added to all wells of 48-MTP to make a total amount of 1 ml. The 48-MTP was incubated at 22 ° C. for 3 to 5 days under only a light period, and determination of frond formation was performed by an inverted microscope. The lowest sample concentration showing frond formation was defined as the minimum effective concentration (MEC), and it was considered that the active substance was concentrated in the sample having the lowest MEC, and thus was used as an index of separation.

5 shows photographs of monostroma oxyspermum culture cells after 5 days of culture when no sample is added. In addition, photographs of monostroma oxyspermum cultured cells 5 days after the culture when the sample was added are shown in FIG. 6. In the presence of the active substance, the formation of the fronds was observed as shown in FIG. 6.

The composition of the ASP7 medium is as follows.

Table 3 : Modified ASP7 Medium (1 L Min, pH 7.8-8.0)

Distilled Water 950 mlNaCl 25 gMgSO ₄ ㆍ 7H ₂ O 9 gKCl 700 mgCaCl ₂ 840 mgTrisHCl 1 gNaNO ₃ 50 mgNa ₂ -glyceroPO ₄ 20 mgNa ₂ SiO ₃ 9H ₂ O 70 mgVitamin B12 1 μg Nitrilotriacetic Acid 70 mgVitamin Mix S3 * 1 10 ml PII metals * 2 30 ml S2 metals * 3 5 ml

Table 4 : * 1 Vitamin Mix S3

Distilled water 100 ml Thiamine hydrochloride 5 mg Nicotinic acid 1 mg calcium pantothenate 1 mgp-aminobenzoic acid 0.1 mg biotin 0.01 mg inositol 50 mg thymine 30 mg folic acid 0.02 mg

Table 5 : * 2 PII metals

Distilled water 1000 mlNa ₂ -EDTA 1 gFeCl ₃ 6H ₂ O 48 mgH ₃ BO ₃ 1.13 gMnCl ₂ 4H ₂ O 144 mgZnCl ₂ 5.2 mgCoCl ₂ 6H ₂ O 4 mg

Table 6 : * 3 S2 metals

Distilled water 500 mlNa ₂ MoO ₄ ㆍ 2H ₂ O 63 mgNaBr 640 mgSrCl ₂ ㆍ 6H ₂ O 304 mgRbCl 14 mgLiCl 61 mgKI 0.65 mgV ₂ O ₅ 0.18 mg

Example 2 Isolation and Purification of New Compound 1

YM-2-23 strain (FERM BP-8417) was used as a seed bacterium. This culture medium was used for marine broth (manufactured by Difco, 37.4g / l or by mixing the reagents according to the display components). The strain was incubated in a 100 ml Erlenmeyer flask with 50 ml of marine broth for 24 hours at 30 ° C (100 revolutions per minute), and then again inoculated with a 1 L baffle flask containing 450 ml of medium. Incubated for 24 hours under the same conditions. The culture was performed by shaking culture (130 revolutions per minute) in 16 1L baffle flasks containing 800 ml of medium and shaking culture (100 rotations per minute) in 10 1L Erlenmeyer flasks containing 450 ml of medium. Was 30 ° C., and the incubation time was 3 days. About 18 L of the culture solution thus obtained was centrifuged. Cells were stored at −20 ° C. and the culture filtrate was stored at 4 ° C. until extraction. Cells of 4 times of the main culture (about 72 L) were extracted twice in 1200 ml of 50% acetonitrile aqueous solution and concentrated. The concentrate was mixed with about 72 L of the culture filtrate and adsorbed onto 2500 ml of Diaion HP-20 (manufactured by Mitsubishi Chemical), a styrenedivinylbenzene polymer. The resin was washed with 6000 ml of 10% acetonitrile aqueous solution and desalted, and eluted with 6000 ml of 50% acetonitrile aqueous solution to obtain a fraction that attracted frond formation. The active fraction was adsorbed on TOYOPEARL DEAE-650 (M) (manufactured by Tohso), washed with 180 mM NaCl-20% acetonitrile solution, and the active fraction was eluted with 450 mM NaCl-20% acetonitrile solution. The elution fractions were concentrated under reduced pressure to remove acetonitrile, and then adsorbed onto 500 ml of Diaion HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.), washed with 1000 ml of 10% acetonitrile solution and desalted, followed by active fraction with 1000 ml of 50% acetonitrile solution. Obtained. After concentration under reduced pressure, lyophilization was performed to obtain a frond attractant fraction. Gel culture chromatography (100 ml NaCl-20 mM Na ₂ HPO ₄ -20% aqueous acetonitrile solution (pH 9) as a mobile phase was mixed by mixing two batches (about 140 L of culture medium) of the electroculture and coarse fraction. Purified by Amersham Bioscience, Sephacryl S-100 HR inner diameter 25mm x length 1200mm) to obtain a fraction showing the frond attractant activity. The active fractions were concentrated, desalted, lyophilized and subjected to high performance liquid chromatography using an aqueous solution of 14 to 22% acetonitrile 5 g (NH ₄ ) ₂ CO ₃ / L (Amersham Bioscience, RESOURCE RPC 3ML, inner diameter 6.4 mm). x 100 mm x 2 in series) yielding about 140 μg of novel compound 1 of the present invention having electrochemical properties.

Example 3 Isolation and Purification of New Compound 2

Novel Compound 1 isolated according to Example 2 has been demonstrated to vary in form of Novel Compound 2 under strong alkaline conditions of 5-25% acetonitrile-1% NH ₃ aqueous solution in the final tablets. In addition, novel compound 1 is present together with novel compound 2 in the culture supernatant before purification. New compound 2 is very unstable at neutral-weak acidity, but new compound 1 derived under alkaline conditions is relatively stable. High-performance liquid chromatography using 17% acetonitrile-5 g (NH ₄ ) ₂ CO ₃ +5 mL NH ₃ / L aqueous solution as the mobile phase in the final purification step of Example 2 (3 mL of RESOURCE RPC, Amersham Bioscience, 6.4) mm x 100 mm x 2 in series), Compound 2 showing a ¹ H-NMR spectrum as shown in FIG. 4 can be obtained.

Example 4 Preparation and Physicochemical Properties of Methylated Compounds of Novel Compound 1 and Derivatives thereof

Approximately 140 µg of the novel compound 1 was dissolved in 40 µl of methanol, and 160 µl of benzene and 100 µl of trimethylsilyldiazomethane (10%, n-hexane solution, manufactured by Tokyo Kasei Kogyo) were added and stirred well. Reacted. A small amount of acetic acid was added little by little until the yellow color of diazomethane disappeared, and the solvent was distilled off and removed by an evaporator, followed by high performance liquid chromatography using an aqueous solution of 50-100% acetonitrile (Tosho, TSKgel ODS-80Ts) When separated by 4.6 mm x 150 mm in length, trimethyl body of novel compound 1 (hereinafter abbreviated as Me1) was obtained almost quantitatively (yield about 152 µg, yield 99%). In addition, the trimethyl body is replaced with methanol (deuterium methanol) methoxy group in one of the methoxy group of slowly (-OCH ₃₎ (-OCD ₃₎ of the NMR measurement solvent provides Me1B. About 152 µg of Me1 was dissolved in 200 µl of methanol, and 1 mg of sodium borohydride was added while cooling with ice, followed by reduction for 1 hour. When the reaction solution was separated by high-performance liquid chromatography using 50-100% acetonitrile aqueous solution as a mobile phase (Tosho, TSKgel ODS-80Ts, inner diameter 4.6mm x length 150mm), high yield (yield 140µg, yield 98) %) To give a reduced form of Me1 (hereinafter abbreviated as Me1H3). The total amount of Me1H3 obtained here was dissolved in 400 µl of dry diethyl ether, and 100 µl of 20 mg / ml sodium borohydride ethanol solution was added at room temperature, followed by reaction at room temperature for 150 minutes. 100 µl of saturated aqueous sodium chloride solution was added and stirred for 10 minutes, and then the solvent was distilled off by an evaporator. When separated by high-performance liquid chromatography using 50-100% acetonitrile aqueous solution as a mobile phase (Tosho, TSKgel ODS-80Ts, inner diameter 4.6mm x length 150mm), it was almost quantitative (yield 123µg, yield 99%). A high reducing body of Me1 (hereinafter abbreviated as Me1H1) was obtained. Again, the entire amount of Me1H1 obtained therein was dissolved in 400 µl of dry dimethyl sulfoxide, 1 mg of finely crushed sodium hydroxide was added, and 40 µl of methyl iodide was added and allowed to react at room temperature for 30 minutes. After stopping the reaction by adding 500 µl of distilled water while cooling with ice, the reaction solution was subjected to high-performance liquid chromatography (Tosho Co., TSKgel ODS-80Ts, inner diameter of 4.6 mm x length 150 mm) using 50-100% aqueous acetonitrile solution as a mobile phase. When isolated as it was, the methylation of Me1H1 (hereinafter abbreviated as Me1H1Me) was obtained almost quantitatively (yield 135 micrograms, yield 99%).

The compound obtained here exhibited the following physicochemical properties.

Physicochemical Properties of Me1

1. Color of material: colorless

2. Molecular Weight: 499

3. Molecular Formula: C ₂₇ H ₃₇ N ₃ O ₄ S

Mass spectrometry: FABMS: m / z 500 [M + H] ⁺

High resolution mass spectrometry: found 500.2569 [M + H] ⁺

Calculated 500.2583 (C ₂₇ H ₃₈ N ₃ O ₄ S)

4. Nuclear magnetic resonance signal:

1) ¹ H-NMR (heavy methanol, 500 MHz): (FIG. 7)

δppm 0.916 (3H, s), 0.941 (3H, s), 0.973 (3H, s), 1.086 (1H, ddd, J = 3.5, 12.5, 13.0 Hz), 1.136 (1H, dd, J = 2.0, 12.0 Hz ), 1.271 (1H, ddd, J = 4.0, 13.0, 14.0 Hz), 1.380 (3H, s), 1.47 (2H, m), 1.50 (1H, m), 1.65 (1H, m), 1.69 (2H, m), 1.73 (1H, m), 1.84 (1H, m), 2.130 (1H, ddd, J = 3.5, 7.0, 12.5 Hz), 2.34 (2H, m), 3.488 (3H, s), 3.914 (3H , s), 4.034 (3H, s), 7.911 (1H, d, J = 8.0 Hz), 8.258 (1H, d, J = 8.0 Hz)

2) ¹³ C-NMR (heavy methanol, 125 MHz): (FIG. 8)

δ ppm 15.413 (q), 19.559 (t), 20.797 (t), 20.961 (q), 21.981 (q), 28.416 (t), 33.855 (q), 34.164 (s), 37.928 (s), 40.356 (t) , 41.722 (t), 42.996 (t), 52.266 (q), 53.255 (q), 52.255 (q), 53.328 (d), 57.432 (d), 79.291 (s), 120.0 (s), 127.392 (d) , 140.407 (d), 141.6 (s), 146.659 (s), 149.8 (s), 164.761 (s), 166.027 (s), 167.402 (s)

5. Solubility: Poorly soluble in water and DMSO, soluble in aqueous solvents such as 50-100% aqueous methanol solution, 50-100% acetonitrile aqueous solution, poorly soluble in low polar organic solvents such as hexane and chloroform

Physicochemical Properties of Me1B

1. Color of material: colorless

2. Molecular Weight: 502

3. Molecular Formula: C ₂₇ H ₃₄ D ₃ N ₃ O ₄ S

Mass spectrometry: FABMS: m / z 503 [M + H] ⁺

High resolution mass spectrometry: found 503.2777 [M + H] ⁺

Calculated 503.2772 (C ₂₇ H ₃₅ D ₃ N ₃ O ₄ S)

4. Nuclear magnetic resonance signal:

¹ H-NMR (heavy methanol, 500 MHz): (Figure 9)

δppm 0.916 (3H, s), 0.941 (3H, s), 0.973 (3H, s), 1.086 (1H, ddd, J = 3.5, 12.5, 13.0 Hz), 1.136 (1H, dd, J = 2.0, 12.0 Hz ), 1.271 (1H, ddd, J = 4.0, 13.0, 14.0 Hz), 1.380 (3H, s), 1.47 (2H, m), 1.50 (1H, m), 1.65 (1H, m), 1.69 (2H, m), 1.73 (1H, m), 1.84 (1H, m), 2.130 (1H, ddd, J = 3.5, 7.0, 12.5 Hz), 2.34 (2H, m), 3.488 (3H, s), 3.914 (3H , s), 7.911 (1H, d, J = 8.0 Hz), 8.258 (1H, d, J = 8.0 Hz)

5. Solubility: Poorly soluble in water and DMSO, soluble in aqueous solvents such as 50-100% aqueous methanol solution, 50-100% acetonitrile aqueous solution, poorly soluble in low polar organic solvents such as hexane and chloroform

Physicochemical Properties of Me1H3

1. Color of material: colorless

2. Molecular Weight: 471

3. Molecular Formula: C ₂₆ H ₃₇ N ₃ O ₃ S

Mass spectrometry: FABMS: m / z 472 [M + H] ⁺

High resolution mass spectrometry: found 472.2630 [M + H] ⁺

Calc. 472.2634 (C ₂₆ H ₃₈ N ₃ O ₃ S)

4. Nuclear magnetic resonance signal:

1) ¹ H-NMR (DMSO-d6, 500 MHz): (FIG. 10)

δ ppm 0.802 (3H, s), 0.810 (3H, s), 0.885 (3H, s), 0.951 (1H, m), 1.018 (1H, m), 1.150 (1H, m), 1.254 (3H, s), 1.35 (1H, m), 1.36 (1H, m) 1.37 (1H, m), 1.50 (1H, m), 1.54 (1H, m), 1.57 (1H, m), 1.58 (1H, m), 1.688 ( 1H, m), 1.984 (1H, m), 2.174 (2H, br d, J = 8.5 Hz), 3.399 (3H, s), 3.741 (3H, s), 4.570 (2H, br d, J = 5.5 Hz ), 5.495 (1H, brt, J = 5.5 Hz), 7.587 (1H, d, J = 8.0 Hz), 7.722 (1H, d, J = 8.0 Hz)

2) ¹³ C-NMR (calculate chemical shift based on heavy methanol, Heteronuclear Multiple Bond Coherence (HMBC) and Heteronuclear Single Quantum Coherence (HSQC) spectra):

δ ppm 14.2 (q), 17.7 (t), 19.0 (t), 20.0 (q), 21.1 (q), 26.7 (t), 33.0 (q), 33.3 (s), 36.1 (s), 38.3 (t) , 40.1 (t), 41.1 (t), 51.2 (q), 51.2 (d), 52.0 (q), 55.2 (d), 63.7 (t), 76.8 (s), 119.2 (s), 121.8 (d) , 134.1 (s), 138.3 (d), 146.2 (s), 159.8 (s), 162.8 (s), 166.2 (s)

5. Solubility: Soluble in water and 100% methanol, soluble in water-soluble solvents such as 50% methanol aqueous solution, 50% acetonitrile aqueous solution, soluble in DMSO, poorly soluble in low polar organic solvents such as hexane and chloroform

Physicochemical Properties of Me1H1

1. Color of material: colorless

2. Molecular Weight: 415

3. Molecular Formula: C ₂₄ H ₃₇ N ₃ OS

Mass spectrometry: FABMS: m / z 416 [M + H] ⁺

High resolution mass spectrometry: found 416.2747 [M + H] ⁺

Calculated 416.2735 (C ₂₄ H ₃₈ N ₃ OS)

4. Nuclear magnetic resonance signal:

1) 1 H-NMR (heavy chloroform, 500 MHz): (FIG. 11)

δ ppm 0.842 (6H, s), 0.918 (3H, s), 0.97 (1H, m), 1.04 (1H, m), 1.18 (1H, m), 1.327 (3H, s), 1.36 (1H, m), 1.42 (1H, m), 1.45 (1H, m), 1.55 (1H, m), 1.58 (1H, m), 1.64 (1H, m), 1.667 (1H, dd, J = 5.5, 12 Hz), 1.78 (1H, m), 2.02 (2H, m), 2.05 (1H, m), 3.650 (2H, d, J = 12 Hz), 4.647 (2H, d, J = 14 Hz), 4.801 (2H, br s ), 7.226 (1H, d, J = 8.0 Hz), 7.474 (1H, d, J = 8.0 Hz)

2) ¹³ C-NMR (calculate chemical shift based on heavy chloroform, Heteronuclear Multiple Bond Coherence (HMBC) and Heteronuclear Single Quantum Coherence (HSQC) spectra)

δ ppm 15.1 (q), 18.4 (t), 19.8 (t), 20.9 (q), 21.5 (q), 25.1 (t), 33.2 (s), 33.3 (q), 36.8 (s), 39.2 (t) , 40.7 (t), 41.7 (t), 52.5 (d), 56.0 (d), 60.61 (t), 62.33 (t), 64.73 (t), 77.0 (s), 106.0 (s), 119.8 (d) , 133.2 (s), 139.7 (d), 148.0 (s), 156.2 (s)

5. Solubility: Soluble in water, soluble in aqueous solvents such as 10-100% aqueous methanol solution, 10-100% acetonitrile aqueous solution, soluble in low polar organic solvents such as hexane and chloroform.

Physicochemical Properties of Me1H1Me

1. Color of material: colorless

2. Molecular Weight: 457

3. Molecular Formula: C ₂₇ H ₄₃ N ₃ OS

Mass spectrometry: FABMS: m / z 458 [M + H] ⁺

High resolution mass spectrometry: found 458.3201 [M + H] ⁺

458.3205 (C ₂₇ H ₄₄ N ₃ OS)

4. Nuclear magnetic resonance signal:

1) ¹ H-NMR (heavy chloroform, 500 MHz): (FIG. 12)

δppm 0.847 (6H, s), 0.921 (3H, s), 0.96 (1H, m), 1.05 (1H, m), 1.18 (1H, m), 1.331 (3H, s), 1.36 (1H, m), 1.41 (1H, m), 1.43 (1H, m), 1.57 (1H, m), 1.59 (1H, m), 1.67 (1H, m), 1.68 (1H, m), 1.77 (1H, br d, J = 13 Hz), 2.00 (1H, m), 2.09 (1H, m), 2.16 (1H, m), 3.19 (3H, br s), 3.45 (3H, br s), 3.509 (3H, s), 3.564 (2H, d, J = 12 Hz), 4.419 (2H, dd, J = 8.5, 10.5 Hz), 4.644 (2H, br s), 7.356 (1H, d, J = 7.0 Hz), 7.488 (1H, d , J = 7.0 Hz)

2) ¹³ C-NMR (calculate chemical shift based on heavy chloroform, Heteronuclear Multiple Bond Coherence (HMBC) and Heteronuclear Single Quantum Coherence (HSQC) spectra)

δ ppm 15.0 (q), 18.7 (t), 20.0 (t), 20.6 (q), 21.7 (q), 26.2 (t), 33.2 (s), 33.5 (q), 36.9 (s), 39.4 (t) , 41.0 (t), 42.0 (t), 52.8 (d), 56.2 (d), 58.4 (q), 59.0 (q), 59.1 (q), 70.2 (s), 74.0 (s), 75.6 (s) , 76.9 (s), 109.9 (s), 120.5 (d), 135.1 (s), 139.2 (d), 145.7 (s), 155.1 (s)

5. Solubility: Soluble in water, soluble in water solvents such as 30-100% methanol aqueous solution, 30-100% acetonitrile aqueous solution, and soluble in low polar organic solvents such as hexane and chloroform.

Example 5 Examination of Minimum Effective Concentration (MEC) of New Compound 1

In the same manner as in Example 1, new compound 1 isolated and purified at 48-MTP was started at a final concentration of 1 µg / ml and 16 dilutions were performed. At this time, Monostroma oxyspermum cells were about 20 cells per ml of medium. When diluting, the pipette tip was exchanged for each step dilution. Three sets of this series of operations were prepared and cultured for 3 days as in Example 1. As a result, frond formation of Monostroma oxyspermum was observed up to 12 dilutions of heat. From this, the MEC of novel compound 1 was 1 μg / ml x 10 ^-12 = 1ag / ml (atto-gram per milliliter). As organs were cultured, new compound 1 was consumed by proliferated Monostroma oxyspermum , and the fronds began to collapse. The photograph showing the result of 7 steps of dilution after 10 days of culture (final concentration of new compound 1 1 × 10 ⁻⁷ μg / ml) was shown in FIG. 13. The frond collapse was observed, although the frond remained somewhat circular. A photograph showing the result of six dilutions (final concentration 1 × 10 ⁻⁶ μg / ml of new compound 1) after 10 days of culture is shown in FIG. 14. The fronds remained undisrupted and the MEC after 10 days of culture was 1 × 10 ⁻⁶ μg / ml = 1 pg / ml (pico-gram per milliliter). Since monostroma oxyspermum cells perform cell division about twice a day under the conditions of Example 1, when the initial cell number is 20, three days later,

20 x (2 x 2) ³ = 1,280 cells

But in calculation for ten days in calculation,

20 x (2 x 2) ¹⁰ = 20,971,520 cells

After 10 days compared to 3 days, the number of cells multiply by 10,000 times or more, it can be explained that the MEC changes significantly depending on the number of cells and the number of culture days. As a result, new compound 1 needs to be added according to the number of days of culture and the proliferation of cells.

Example 6 Activity of Novel Compound 1 and Derivatives

As in Example 5, the minimum effective concentration of New Compound 1 and its derivatives was measured, and the following results were obtained.

Table 7 : Minimum Effective Concentrations

New Compound 1 10 ^-12 μg / mLMe1 10 ^-4 μg / mLMe1H3 10 ^-2 μg / mLMe1H1 No activity

Methylation of novel compound 1 reduced the activity to 1/10 ⁸ . Therefore, as shown in Example 4, it was considered that the functional group methylated by methylating agents, such as trimethylsilyl diazomethane, is important for the activity.

Example 7 Culture of Ulva pertusa and Enteromorpha intestinalis

Free-living cells obtained from Ulva pertusa and Enteromorpha intestinalis collected from Miho Shimizu City, Shizuoka-ken were washed in the ASP7 medium to which antibiotic mixture was added using luminescence, followed by a square petri dish filled with sterile cover glass. Addition was carried out for 5 days of aseptic treatment. When the jujube cells adhered to the cover glass and started to grow, each cover glass was placed in each well of 6-MTP, and 10 ml of ASP7 medium to which the antibiotic mixture was not added was added. New compound 1 was added to the experimental group to 1 ng / ml, and nothing was added to the control group, and cultured under the same conditions as in Example 1 for 7 days. After 10 days, the cover glass of the test and control groups were transferred to the culture test tube to which the medium under the same conditions was added and cultured again for 7 days. Experimental results of Ulva pertusa are shown in FIG. 15, and experimental results of Enteromorpha intestinalis are shown in FIG. 16. As can be seen from the comparison of the experimental and control groups, only the rhizooids developed abnormally, and no normal frond formation was observed, but the addition of new compound 1 resulted in normal development and frond formation. Could see.

In addition, in the ASP7 medium to which the antibiotic mixture is added, 2% of the antibiotic mixture having the following composition is added to the ASP7 medium.

Table 8 : Antibiotic Mixtures

Distilled water 1,000 ml Penicillin 100 mg Streptomycin 200 mg Kanamycin 100 mg

Example 8 Specific Activity of Morphogenesis Induction for Large Green Algae

In the same manner as in Example 5, the minimum effective concentrations of the YM-1-69 strain, the YM-2-23 strain and the similar strains shown in FIG. 17 were measured. As a result, the same results as in FIG. 18 were obtained. Here, the horizontal axis represents the number of ml of Monostroma oxyspermum culture activated with 1 µl of the culture supernatant of similar strains. For example, the YM-2-23 strain in FIG. 18 may activate about 8,000 ml of Monostroma oxyspermum culture with 1 µl of the culture supernatant.

Example 9 Determination of Substructures of New Compound 1 and Derivatives thereof

From the various NMR spectra and mass spectra of the novel compound 1, the partial structure shown in FIG. 19 was derived. By analyzing the HMBC spectrum of these substructures, the driman type telephenoid structure and the tetrasubstituted benzene ring shown in FIG. 20 can be connected. As shown in FIG. 21, from the NMR spectrum of the trimethylated Me1 of the novel compound 1, the relative stereoscopic arrangement was determined by analyzing the partial structure and the NOESY spectrum of the monosubstituent of the tetrasubstituted benzene ring. Moreover, as shown in FIG. 22, the estimation structure so far was confirmed from the HMBC spectrum. As shown in Figs. 23 and 24, in Me1H3, correlation peaks of NOESY and HMBC spectra were observed between the driman moiety and the benzene ring, and the structures of the two were represented by carbon numbers 11 to 16, and 8-O-21. The substructure was estimated by In the reduced compound Me1H1 of the novel compound 1, a correlation peak is observed between the methylene group at the 24th position and the 21st position on the benzene ring, As shown in FIG. 25 and FIG. 26, the last substituent of the tetrasubstituted benzene ring was determined. From various NMR spectra of the methylated Me1H1Me of Me1H1, as shown in Figs. 27 and 28, the correlation between the benzene ring, the N-methyl group, and the methylene group at the 24th position was observed, and the estimated partial structure so far was confirmed.

As shown in Example 4, Me1H1Me is a compound obtained by methylation, reduction and remethylation of novel compound 1, and novel compound 1 has the same structure as in Chemical Formula 1 below.

Formula 1 :

In addition, as shown in Example 3, since the new compound 1 is a derivative by alkali treatment of the new compound 2, the new compound 2 is represented by any of Formula 2, Formula 3 or Formula 4, which is a ketone body of the compound of Formula 1 Compounds or mixtures thereof.

Formula 2 :

Formula 3 :

Formula 4 :

In addition, the structure of Me1 is represented by Formula 5, the structure of the heavy methoxy substituent Me1B of Me1 disclosed in Example 4 is represented by Formula 6, the structure of the partially reduced Me1H3 by Me1 is represented by Formula 7, the structure of Me1H1 is reduced by Me1 To the formula (8) is shown in the formula (9) the structure of the re-methylated Me1H1Me of Me1H1.

Formula 5 :

Formula 6 :

Formula 7 :

Formula 8 :

Formula 9 :

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

The present invention provides a novel culture and culture technology for marine large green algae such as Ulva lactuca and Monostroma nitidum using the novel compounds. According to this method, as well as the safe maintenance of aseptic culture and seedling in the laboratory, it is possible to perform the safe growth and production of marine large green algae in aquaculture sites.

<110> MARINE BIOTECHNOLOGY INSTITUTE CO., LTD <120> Novel Chemical Substance Having Morphogenetic and Growth-Accelerating Activities <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 190 <212> DNA <213> Tenacibaculum sp. <400> 1 cctacgggag gcagcagtga ggaatattgg tcaatggagg caactctgaa ccagccatgc 60 cgcgtgtagg aagactgccc tatgggttgt aaactacttt tatatgggaa gaaacccctc 120 ttacgtgtag aggcttgacg gtaccataag aataagcacc ggctaactcc gtgccagcag 180 ccgcggtaat 190 <210> 2 <211> 189 <212> DNA <213> Unknown <220> <223> DNA nucleotide sequence of V3 region in 16S rRNA of YM-2-23 strain being identified as Cytophaga-Flavobacterium-Bacteriodes complex <400> 2 cctacgggag gcagcagtga ggaatattgg acaatgggcg ggagcctgat ccagccatgc 60 cgcgtgcggg aagaaggccc tatgggtcgt aaaccgcttt tatacgggaa gaaaccaccc 120 tacgtgtagg gtactgacgg taccgtaaga ataaggaccg gctaactccg tgccagcagc 180 cgcggtaat 189 <210> 3 <211> 1173 <212> DNA <213> Tenacibaculum sp. <400> 3 gtatctggag gtttacacgg agttggtgta tcttgtgtga atgcactttc agatcattta 60 aaagctacag ttcacagaga aggtaaaata tgggaacaag agtatgaacg tggtaaaaca 120 ctttatcctg taaaaactgt aggtgaaact gatataactg gtacagaagt aactttctta 180 ccagacaaaa gtattttcca acaaaccaca gaatataatt acgaaacgtt agctacacgt 240 atgcgtgagt tagcgtatct taataaagga atcacgatta cgttaacaga taagcgtaat 300 aaagatgatg aaggaaattt tattgctgaa actttccaca gtaacgaagg attatctgaa 360 tttgttaaat atttagatag tactcgtact cctgttattc agcatgtaat ttcaatggaa 420 ggtgagaaaa acggaattcc tgttgaggtt gcaatgattt ataatgattc atatgctgaa 480 aatttacatt cttatgtaaa taacattaat actcacgaag gaggaacaca tttatcagga 540 tttagaagag gtttaacaag tactttaaag aaatatgcag atacttctgg attactaaag 600 aacgttaagt ttgagatttc tggagatgat ttccgtgaag gtttaacggc aattgtatct 660 gtaaaagtag ctgaacctca gtttgaagga caaacaaaaa caaaattagg aaacagagaa 720 gttacttctg cagtatcgca agctgtagca gaaatgttaa ctgattattt agaggaaaat 780 cctaatgatg ctaaaacgat tgtacaaaaa gtaattcttg cagctcaagc gcgtcacgca 840 gctcgtaaag caagagaaat ggtgcaacgt aaaacagtaa tgagtattgg aggtttacct 900 ggtaaactat ctgattgttc tgaaactgat ccagcagttt gtgaaatttt cttagtcgag 960 ggagattcgg caggtggaac tgcaaaacaa ggtcgtgatc gtaatttcca agcaatttta 1020 cccttacgtg gtaagattct taacgtagaa aaagcgatgc agcataaagt ttttgagaat 1080 gaagaaatca aaaacatgtt tacggcttta ggaatcacta tcggaacaga agaagatcca 1140 agagcattaa acttatcaaa attaagatat cat 1173 <210> 4 <211> 1173 <212> DNA <213> Unknown <220> <223> DNA nucleotide sequence of gyrB gene of YM-2-23 strain being identified as Cytophaga-Flavobacterium-Bacteriodes complex <400> 4 gtatccggtg gtttgcacgg ggtaggtgtt tcttgtgtga acgccctttc caatcatctt 60 aaagctaccg tacatagaga tgggaaagtt tgggaacagg aatatgaacg gggtaaatcc 120 ctttatcccg taaaaagtgt tggggagacc gatgaaactg gaaccattgt taccttcata 180 ccagatgatt caatctttac ccaaacaaca gagtatagtt atgagaccct tgccaacaga 240 atgcgtgagc tttcgttctt gaacaaaggg gttaccatta gcattacgga caaaagagta 300 aaggataaag aaggggagta cctttctgaa actttttatt ccgatgctgg actaagtgaa 360 tttgttaagt tcttggatgg tacccgtgaa cctttgattc aaggggttat cgcgatggaa 420 ggggagaaaa atggtatccc tgtggaagtg gcaatggttt acaacaccag ttacacggag 480 aatttacatt cctatgtgaa taacattaac acgcacgaag ggggtacgca tctttccggt 540 tttagaaggg gattgacctc tactttaaag aaatacgcag attcttctgg aatgctcgag 600 aaattgaagt ttgaggttca gggagatgat ttccgtgaag gacttacagc aattgtttcc 660 gttaaggtcg cagaacctca atttgaaggt cagacgaaaa ccaagcttgg aaaccgcgag 720 gtttcttctg cggtgagcca agctgtttct gaaatgctca cggattattt ggaggagcat 780 ccagatgatg ccaaggttat tgttcaaaaa gttatccttg ccgctcaggc cagacatgcc 840 gctacaaagg cccgtgaaat ggtacagcgt aagacggtaa tgagtattgg tgggctacct 900 ggaaaattgt ccgattgttc tgagcaagat cctgcgcaat gtgaagtatt tcttgtagag 960 ggagattctg caggtggtac ggcaaaaatg ggccgggacc gaaaatttca ggccattctt 1020 ccactaaggg gtaaaatctt gaacgtggaa aaagccatgc agcacaaggt ttttgaaaat 1080 gaggaaataa agaatattta tacggcccta ggggttacta ttggaacgga agaagatagt 1140 aaggccttga acctggaaaa attaagatat cat 1173

Claims (10)

Compound 1 having the following physicochemical properties: (i) Color of substance: colorless      (ii) molecular weight: 457 (iii) Molecular Formula: C ₂₄ H ₃₁ N ₃ O ₄ S Mass spectrometry: FABMS: m / z 456 [M ⁻ H] ⁻ (FIG. 1) High resolution mass spectrometry: found 456.1960 [MH] ^- Calculated 456.1930 (C ₂₄ H ₃₁ N ₃ O ₄ S)      (iv) nuclear magnetic resonance signals: 1) ¹ H-NMR (D ₂ O-20 mM Na ₂ HPO ₄ (pH 9), 750 MHz): (FIG. 2) δppm 0.818 (3H, s), 0.837 (3H, s), 0.882 (3H, s), 0.960 (1H, m), 1.058 (1H, m), 1.167 (1H, m), 1.326 (3H, s), 1.37 (1H, m), 1.38 (1H, m), 1.40 (1H, m), 1.58 (1H, m), 1.61 (2H, m), 1.52 (1H, br d, J = 13 Hz), 1.76 (1H , br d, J = 14 Hz), 2.024 (1H, m), 2.181 (1H, dd, J = 4, 14 Hz), 2.291 (1H, dd, J = 14, 16.5 Hz), 7.698 (1H, d, J = 7.5 Hz), 7.845 (1H, d, J = 7.5 Hz) 2) ¹³ C-NMR (D ₂ O-20 mM Na ₂ HPO ₄ (pH 9), 125 MHz): (FIG. 3) δ ppm 15.236 (q), 19.037 (t), 20.287 (t), 20.955 (q), 21.835 (q), 25.987 (t), 33.381 (s), 33.636 (q), 37.308 (s), 39.590 (t) , 41.199 (t), 42.346 (t), 52.769 (d), 56.381 (d), 79.096 (s), 114.965 (s), 124.399 (d), 139.004 (s), 141.232 (d), 150.282 (s) , 152.656 (s), 172.081 (s), 173.538 (s), 174.661 (s). Compound 2 having the following chemical properties: (i) Color of substance: colorless      (ii) nuclear magnetic resonance signals: ¹ H-NMR (D ₂ O-20 mM Na ₂ HPO ₄ (pH 9), 500 MHz): (Figure 4) δ ppm 0.815 (3H, s), 0.834 (3H, s), 0.877 (3H, s), 0.949 (1H, m), 1.048 (1H, m), 1.163 (1H, m), 1.297 (3H, s), 1.35-1.40 (3H, m), 1.52-1.63 (4H, m), 1.753 (1H, br d, J = 14 Hz), 2.012 (1H, m), 2.158 (1H, m), 2.299 (1H, m) , 7.646 (1H, d, J = 8.0 Hz), 7.769 (1H, d, J = 8.0 Hz). A method for producing Compound 1, comprising culturing the microorganism having the ability to produce Compound 1 in a medium, and producing and accumulating Compound 1 in a culture to obtain Compound 1, which is generated and accumulated. A method for producing compound 2, wherein the microorganism having the ability to produce compound 2 of claim 2 is cultured in a medium, and compound 2 is produced and accumulated in the culture, thereby obtaining compound 2, which is generated and accumulated. The method of claim 3, wherein The microorganism is characterized in that the strain YM-2-23 (FERM BP-8417), Tenacibaculum sp. YM-1-69 (FERM BP-8418) or their similar strains Preparation of Compound 1. The method of claim 4, wherein The microorganism is characterized in that the strain YM-2-23 (FERM BP-8417), Tenacibaculum sp. YM-1-69 (FERM BP-8418) or their similar strains Preparation of Compound 2. Algae culture medium comprising the compound of claim 1 as an active ingredient. Algae culture medium comprising the compound of claim 2 as an active ingredient. A monomethylated, dimethylated or trimethylated compound of Compound 1 obtained by treating compound 1 of claim 1 with trimethylsilyldiazomethane. A compound obtained by treating the trimethylated product of claim 9 with sodium borohydride or a derivative thereof.